During glycolysis, cells convert simple sugars like glucose into pyruvic acid, with a net production of ATP and NADH. In the absence of a functioning electron transport system for oxidative phosphorylation, at least 95% of the pyruvic acid is consumed in short pathways which regenerate NAD.sup.+, an obligate requirement for continued glycolysis and ATP production. The waste products of these NAD.sup.+ regeneration systems are commonly referred to as fermentation products.
Microorganisms are particularly diverse in the array of fermentation products which are produced by different genera. These products include organic acids, such as lactate, acetate, succinate and butyrate, as well as neutral products such as ethanol, butanol, acetone and butanediol. Indeed, the diversity of fermentation products from bacteria has led to their use as a primary determinant in taxonomy. See, for example, BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY, Williams & Wilkins Co., Baltimore (1984) (hereafter "Bergey's Manual").
End products of fermentation share several fundamental features. They are relatively nontoxic under the conditions in which they are initially produced but become more toxic upon accumulation. They are more reduced than pyruvate because their immediate precursors have served as terminal electron acceptors during glycolysis. The microbial production of these fermentation products forms the basis for our oldest and most economically successful applications of biotechnology and includes dairy products, meats, beverages and fuels. In recent years, many advances have been made in the field of biotechnology as a result of new technologies which enable researchers to selectively alter the genetic makeup of some microorganisms.
The bacterium Escherichia coli is an important vehicle for the cloning and modification of genes for biotechnology, and is one of the most important hosts for the production of recombinant products. In recent years, the range of hosts used for recombinant DNA research has been extended to include a variety of bacteria, yeasts, fungi, and eukaryotic cells. The invention described here relates to the use of recombinant DNA technology to elicit the production of specific useful products by a modified host.
The DNA used to modify the host of the present invention can be isolated from Zymomonas mobilis, a bacterium which commonly is found in plant saps and in honey. Z. mobilis has long served as an inoculum for palm wines and for the fermentation of Agave sap to produce pulque, an alcohol-containing Mexican beverage. The microbe also is used in the production of fuel ethanol, and reportedly is capable of ethanol-production rates which are substantially higher than that of yeasts.
Although Z. mobilis is nutritionally simple and capable of synthesizing amino acids, nucleotides and vitamins, the range of sugars metabolized by this organism is very limited and normally consists of glucose, fructose and sucrose. Substrate level phosphorylation from the fermentation of these sugars is the sole source of energy for biosynthesis and homeostasis. Zymomonas mobilis is incapable of growth even in rich medium such as nutrient broth without a fermentable sugar.
Z. mobilis is an obligatively fermentative bacterium which lacks a functional system for oxidative phosphorylation. Like the yeast Saccharomyces cerevisiae, Z. mobilis produces ethanol and carbon dioxide as principal fermentation products. Z. mobilis produces ethanol by a short pathway which requires only two enzymatic activities: pyruvate decarboxylase and alcohol dehydrogenase. Pyruvate decarboxylase is the key enzyme in this pathway which diverts the flow of pyruvate to ethanol. Pyruvate decarboxylase catalyzes the nonoxidative decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. Two alcohol dehydrogenase isozymes are present in this organism and catalyze the reduction of acetaldehyde to ethanol during fermentation, accompanied by the oxidation of NADH to NAD.sup.+. Although bacterial alcohol dehydrogenases are common in many organisms, few bacteria have pyruvate decarboxylase. Attempts to modify Z. mobilis to enhance its commercial utility as an ethanol producer have met with very limited success.
Most fuel ethanol is currently produced from hexose sugars in corn starch or cane syrup utilizing S. cerevisiae or Z. mobilis. But such sugars are relatively expensive sources of biomass sugars and have competing value as foods. Starch and sugars represent only a fraction of the total carbohydrates in plants. The dominant forms of plant carbohydrate in stems, leaves, hulls, husks, cobs and the like are the structural wall polymers, cellulose and hemicellulose. Hydrolysis of these polymers releases a mixture of neutral sugars which include glucose, xylose, mannose, galactose, and arabinose. No single organism has been found in nature which can rapidly and efficiently metabolize all of these sugars into ethanol or any other single product of value.
The genes coding for alcohol dehydrogenase II and pyruvate decarboxylase in Z. mobilis have been separately cloned, characterized, and expressed in E. coli. See Brau & Sahm [1986a] Arch. Microbiol. 144: 296-301, [1986b] Arch. Microbiol. 146: 105-10; Conway et al. [1987a] J. Bacteriol. 169: 949-54; Conway et al. [1987b] J. Bacteriol. 169: 2591-97; Neale et al. [1987] Nucleic Acid. Res. 15: 1753-61; Ingram & Conway [1988] Appl. Environ. Microbiol. 54: 397-404; Ingram et al. [1987] Appl. Environ. Microbiol. 53: 2420-25.
Brau and Sahm [1986a], supra, first demonstrated that ethanol production could be increased in recombinant E. coli by the over-expression of Z. mobilis pyruvate decarboxylase although very low ethanol concentrations were produced. Subsequent studies extended this work by using two other enteric bacteria, Erwinia chrysanthemi and Klebsiella planticola, and thereby achieved higher levels of ethanol from hexoses, pentoses, and sugar mixtures. See Tolan & Finn [1987] Appl. Environ. Microbiol. 53: 2033-38, 2039-44.
When a feedstock of simple sugars is available, therefore, the aforementioned microbes generally are useful. Nevertheless, the majority of the world's cheap, renewable source of biomass is not found as monosaccharides but rather in the form of lignocellulose, which is primarily a mixture of cellulose, hemicellulose, and lignin. Cellulose is a homopolymer of glucose, while hemicellulose is a more complex heteropolymer comprised not only of xylose, which is its primary constituent, but also of significant amounts of arabinose, mannose, glucose and galactose. It has been estimated that microbial conversion of the sugar residues present in waste paper and yard trash from U.S. landfills could provide over ten billion gallons of ethanol. While microorganisms such as those discussed above can ferment efficiently the monomeric sugars which make up the cellulosic and hemicellulosic polymers present in lignocellulose, the development of improved methods for the saccharification of lignocellulose remains a major research goal.
Current methods of saccharifying lignocellulose include acidic and enzymatic hydrolyses. Acid hydrolysis usually requires heat and presents several drawbacks, however, including the use of energy, the production of acidic waste, and the formation of toxic compounds which can hinder subsequent microbial fermentations. Enzymatic hydrolysis thus presents a desirable alternative. For example, enzymes can be added directly to the medium containing the lignocellulosic material.
Genetic-engineering approaches for the addition of saccharifying traits to microorganisms for the production of ethanol or lactic acid have been directed at the secretion of high enzyme levels into the medium. That is, the art has concerned itself with modifying microorganisms already possessing the requisite proteins for transporting cellularly-produced enzymes into the fermentation medium, where those enzymes can then act on the polysaccharide substrate to yield mono- and oligosaccharides. This approach has been taken because the art has perceived difficulty in successfully modifying organisms lacking the requisite ability to transport such proteins.